Protein detecting device

ABSTRACT

A protein detecting device, which comprises: ( 1 ) a detecting unit having a bonding section, which has properties for specifically bonding to a protein to be detected, a detecting section for detecting the bonding of the protein to be detected to the bonding section, the detecting section being made up of a polynucleotide double strand and a charge separating group, and an electrode section detecting the change in electrical conductivity of, or amount of transferred charge in, the polynucleotide double strand modified by the bond of the protein, ( 2 ) a standard electrode, ( 3 ) a reference electrode, ( 4 ) a container for housing the detecting unit, the standard electrode and the reference electrode, and containing a sample solutions comprising the protein to be detected, and ( 5 ) a measuring unit for measuring the protein based on a signal detected in the detecting unit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the priority of JapanesePatent Application Nos. 2001-338318, filed on Nov. 2, 2001; 2002-83991,filed on Mar. 25, 2002; and 2002-96165, filed on Mar. 29, 2002, thecontents thereof being incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a device for detecting/determining a protein orproteins and, more specifically, to a device capable of thedetection/determination of a protein or proteins without labeling them.

2. Description of the Related Art

The human genome project, developed in 1990s, was an attempt for somecountries to share decoding of all of the human genetic code. It hasbeen announced that a draft was finished in the summer of 2000. It isexpected that, as functional genome science and structural genomescience progress after this, it will be revealed what function each ofthe decoded human genome sequence data pertains to.

The human genome project has brought a large change in paradigm toscientific technology and industries involving life sciences. Forexample, diabetes mellitus has been classified based on symptom of highblood sugar level and, with respect to the cause of onset, has beenclassified, based on a level of capacity of producing insulin in apatient's body, into type I (insulin cannot be produced in the body) andtype II (the amount of insulin cannot be controlled in the body). Thehuman genome project presented all the data of amino-acid sequence ofproteins, such as enzymes and receptors, pertaining to the control ofthe detection of blood glucose and insulin, or synthesis, decompositionand the like of insulin, and a DNA sequence of genes pertaining to thecontrol of the amounts of such proteins exists. Using such data,diabetes mellitus, a phenomenon of the control of blood glucose levelbeing not functioned, can be classified into subtypes, depending on whatproteins pertaining to the process of the synthesis, decomposition andthe like of insulin are upset, and, accordingly, it must become possibleto carry out an appropriate diagnosis and cure. Particularly,development of new drugs based on the genome data, in which drugs aredeveloped for particular proteins based on the human genome sequence, isbeing energetically promoted by the pharmaceutical industry, and it isexpected that the relief and cure of a symptom will be effected byunderstanding the conditions of a sequence of proteins which arefunctionally related to each other for the symptom and by administeringa genetically developed drug.

To make this possible, a technique enabling simple measurement of theamounts of a sequence of proteins, which are functionally related toeach other, is needed. However, such a technique is being developed as atechnique of analyzing proteome. As a currently established method,there is known a method in which measurement is carried out by combiningtwo-dimensional electrophoresis and mass spectrometric analysis, whichrequires a relatively large-scale apparatus. To determine a patient'ssymptoms at a clinical site, such as a laboratory or at the bedside in ahospital, the development of a simple, novel technique is needed.

A so-called DNA chip is designed to be adapted for the determination ofa DNA in a sample to be detected by previously introducing thereinto afluorescent pigment during the amplification (increment) thereof by aPCR (polymerase chain reaction), and determining the amount of DNAbonded to complementary DNA chains arranged on a chip in the form ofarray by the intensity of fluorescence. In contrast, proteins cannot beprocessed by what corresponds to amplification by the PCR reaction, asin the case of DNA. Also, there has been a problem that when pluralkinds of proteins are mixed and present in a sample, uniformlyintroducing a fluorescence label into them cannot be used because thereactivities between the fluorescent pigment and the individual proteinsare different.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a device enabling specificdetermination of a biopolymer or biopolymers, such as proteins, withoutproviding the biopolymer or biopolymers to be detected with afluorescence label or the like.

It is also an object of the invention to provide a technique, whichserves as a constituent technique applicable to a so-called array-chiptechnology, for obtaining data useful in the point of view of proteomeperceiving biopolymers as a mass.

In short, according to the invention, plural kinds of proteins in asample are detected and determined together by arranging, in a form ofarray, antibodies or derivatives thereof having an affinity to a proteinto be detected, and based on correspondences between signals generatedby the bond of the proteins to them and the location thereof in thearray.

The protein detecting device according to a first aspect of theinvention comprises: (1) a detecting unit having a bonding section,which has properties for specifically bonding to a protein to bedetected, a detecting section for detecting the bonding of the proteinto be detected to the bonding section, the detecting section being madeup of a polynucleotide double strand and a charge separating group, andan electrode section picking up the change in electrical conductivity ofor amount of transferred charge in the polynucleotide double strandmodified by the bond of the protein, (2) a standard electrode, (3) areference electrode, (4) a container for housing the detecting unit, thestandard electrode and the reference electrode, and containing a samplesolutions comprising the protein to be detected, and (5) a measuringunit for measuring the protein based on a signal detected in thedetecting unit.

The protein detecting device according to a second aspect of theinvention comprises: (1) a detecting unit having a bonding section,which has properties for specifically bonding to a protein to bedetected, a sensing section made up of a polynucleotide double strandand a fluorescent pigment group, a detecting section for detecting thebonding of the protein to be detected to the bonding section, thedetecting section being made up of a quenching pigment group, and acontrolling section for setting the conformation of the sensing sectionat an initial state, the controlling unit being made up of a pair ofelectrodes, (2) a container for housing the detecting unit, andcontaining a sample solution comprising the protein to be detected, (3)an electric power supply connected to the electrodes of the controllingsection, (4) a source of exciting light for exciting the fluorescentpigment group in the sensing section to generate fluorescence, and (5) aunit for measuring the fluorescence.

The protein detecting device according to a third aspect of theinvention comprises: (1) a detecting unit having a bonding section,which has properties for specifically bonding to a protein to bedetected, a detecting section for detecting the bonding of the proteinto be detected to the bonding section, the detecting section being madeup of a polynucleotide double strand and a light emitting group, and anelectrode section to the surface of which the detection section isanchored, (2) a container for housing the detecting unit, and containinga sample solutions comprising the protein to be detected, and (3) a unitfor measuring emitted light.

In another aspect, the invention provides a biopolymer detecting devicecomprising one or more pairs of electrodes provided with a member(detecting member) having a site (bonding site) capable of being bondedto a biopolymer to be detected, and generating an electrical signal as adetection signal for the biopolymer, a sample solution-holding sectionfor holding a sample solution containing the biopolymer to be detectedbetween the electrodes, and an electrical circuit for processing theelectrical signal from the electrode.

The biopolymer detecting device of this aspect detects and determinesthe biopolymer to be detected, based on the difference in electricalsignal from the electrode before and after filling the sample holdingsection with the sample solution, which difference is caused by thebiopolymer to be detected being bonded to the bonding site which thedetecting member on the electrode has. For the detection/determination,a calibration curve, which has been previously prepared, is used. Thedetection/determination of the biopolymer is possible even when only onepair of electrodes is used. In the case of a plurality of pairs ofelectrodes, more precise detection/determination is possible.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and advantages of the invention will be wellunderstood and appreciated by a person with ordinary skill in the art,from consideration of the following detailed description made byreferring to the attached drawings, wherein:

FIG. 1A schematically shows a response observed as electrical conductionthrough a polynucleotide double strand relative to time in the casewhere a protein to be detected is not bonded to a bonding section at theend of the polynucleotide double strand,

FIG. 1B schematically shows a response observed as electrical conductionthrough a polynucleotide double strand relative to time in the casewhere a protein to be detected is bonded to a bonding section at the endof the polynucleotide double strand,

FIG. 2A schematically shows a response observed as the reduction ofintensity of fluorescence emitted by a fluorescent pigment present atone end of a polynucleotide double strand relative to time in the casewhere a protein to be detected is not bonded to a bonding section at theend of the polynucleotide double strand,

FIG. 2B schematically shows a response observed as the reduction ofintensity of fluorescence emitted by a fluorescent pigment present atone end of a polynucleotide double strand relative to time in the casewhere a protein to be detected is bonded to a bonding section at the endof the polynucleotide double strand,

FIG. 3A schematically shows the intensity of emission from a lightemitting group and the amount of current running from the light emittinggroup to an electrode through a polynucleotide in the case where anobjective protein is not captured,

FIG. 3B schematically shows the intensity of emission by a lightemitting group and the amount of current running from the light emittinggroup to an electrode through a polynucleotide in the case where anobjective protein is captured,

FIG. 4 schematically illustrates a detecting unit used in proteindetecting devices according to the first and third aspects of theinvention,

FIG. 5 schematically illustrates the entire construction of a measuringapparatus using the protein detecting device of Example 1,

FIG. 6 schematically illustrates a detecting unit used in the proteindetecting device of Example 2,

FIG. 7, schematically illustrates the entire construction of a measuringapparatus using the device comprising the detecting unit shown in FIG.6,

FIG. 8 schematically illustrates a measuring apparatus using the proteindetecting device of Example 3,

FIGS. 9A to 9G illustrate the process for manufacturing the biopolymerdetecting device described in Example 4,

FIG. 10 is an schematically enlarged section of the samplesolution-holding section of the biopolymer detecting device manufacturedin Example 4,

FIG. 11 illustrates the biopolymer detecting device of Example 5,

FIG. 12 illustrates the measuring system used in Example 5, and

FIG. 13 shows a flow chart indicating the procedure for thedetermination of a protein to be detected.

DETAILED DESCRIPTION OF THE INVENTION

The detecting unit in the protein detecting device according to theinvention corresponds to a so-called protein chip, and is designed todetect, for example, the decline or enhance of part of a series ofprotein interaction network function starting from insulin receptor andending with glycogen catabolic enzyme in the case where hepatocytehaving diabetes changes intracellular metabolism of glycogen dependenton a state of insulin acceptance. By the use of this technique, itbecomes possible to know the population of proteins, including aso-called posttranslational modification such as a phosphorylation or anaddition of a sugar chain. Consequently, instead of detecting diabetesmellitus on the basis of symptom as before, it becomes possible to knowthat the decline in function of a certain protein pertaining to theinteraction network causes incompetence of glucose metabolism, andappropriate diagnosis and cure corresponding to the cause ofdisfunction, as well as verification of a therapeutic result, becomepossible.

Of course, a similar technique is applicable to the entirety ofmultifactorial disease, such as hypertension, hyperlipidemia, cancer(incompetence of control of cell proliferation) or the like, in additionto diabetes mellitus.

The protein detecting device according to the first aspect of theinvention uses, as the protein detecting unit, a unit having a bondingsection, which has properties for specifically bonding to a protein tobe detected, a detecting section for detecting the bonding of theprotein to be measured to the bonding section, the detecting sectionbeing made up of a polynucleotide double strand and a charge separatinggroup, and an electrode section picking up the change in electricalconductivity of, or amount of transferred charge in, the polynucleotidedouble strand modified by the bond of the protein.

The bonding section in this detecting unit is made up of an antibodywhich specifically bonds to a protein to be detected, or to a fragmentof such an antibody obtained by limitedly decomposing the antibody by,for example, a protease, or an organic compound or biopolymer having anaffinity to a protein to be detected. As the antibody, a monoclonalimmunoglobulin IgG antibody can be used, for example. Also, as afragment derived from an IgG antibody, a Fab fragment of an IgG antibodycan be used, for example. In addition, a piece derived from such a Fabfragment can be also used. Examples of organic compounds having anaffinity to a protein to be measured include enzyme substrate analogues,such as adenosine-5′-O-(3-thiotriphosphate) (also called ATP-gamma-S),enzyme activity inhibitors, neurotransmission inhibitors (antagonists)and the like. As examples of biopolymers having an affinity to a proteinto be detected, proteins representing substrates or catalysts for theprotein to be detected, elementary proteins forming a molecular complexwith the protein to be detected, can be enumerated. In the case where asubstance, such as one described above, forming the bonding sectioncannot be directly linked and fixed to a polynucleotide double strandforming the detecting section, it may be fixed through a linking moiety(typically a divalent group) useful for the linkage.

The detecting section is formed as a nano-structure made up of apolynucleotide double strand and a charge separating group. One end ofthe polynucleotide double strand is linked to the bonding section asdescribed above, and the other end is connected to an electrodedescribed later. The charge separating group may be added to thepolynucleotide double strand by covalent bonding, or may be containedwithin the structure of the polynucleotide double strand (interpositionbetween adjacent complementary moieties (intercalation)), or may beincorporated within a nucleotide chain forming the polynucleotide doublestrand by replacing a part thereof. The charge separating group ispreferably present in the vicinity of the opposite end of the electrode,i.e., the end of the polynucleotide double strand for the bondingsection.

A polynucleotide double strand has electrical conductivity in thelongitudinal direction of the strand due to a pi-electron stack ofaromatic rings of bases forming nucleotides. On the other hand, a chargeseparating group in the invention combined with such a polynucleotidedouble strand is a group causing charge separation when subjected toaction of a potential difference, excited light, or the like, and may bea compound when contained within a polynucleotide double strand asdescribed above. Specifically, the charge separating group is a groupwhich is subjected to withdrawal of electron by a reagent (chargeacceptor, e.g., a ferrocyanide ion referred to below) in a samplesolution to generate a positive hole, a group generating an electron anda positive hole by light excitation, or the like. Either of the electronor the hole generated from a charge separating group by the chargeseparation contributes to electrical conduction through thepolynucleotide double strand, which can be detected by an electrodeconnected to one end of the polynucleotide double strand. In otherwords, in the invention, electrical conductivity through thepolynucleotide double strand is detected in the state in which thecharge separating group is caused to be charge-separated. As the chargeseparating group, porphyrin ferrocenated or the like can be used.

When an electron and a hole are generated from a charge separatinggroup, as in the case of the use of light excitation, the electron orthe hole not pertaining to the electrical conduction through apolynucleotide double strand is received by a charge acceptor present ina sample solution. As the charge acceptor, an anion, such asferrocyanide ion, can be used. When such an anion M^(m−) is used, andthe anion after receiving a charge is represented by M^(n−), m issmaller than n in the case of the anion receiving an electron, and m islarger than n in the case of the anion receiving a hole. When a cationis used as the charge acceptor, there is an opposite relationship.

In the invention, the change in the charge transfer state of thepolynucleotide double strand modified by the bond of a protein to thebonding section at one end of the polynucleotide double strand, i.e.,the change in the electrical conductivity or the amount of transferredchange, is detected by the electrode to which the other end of thedouble strand is connected. The electrical conductivity developed alongthe direction of longitudinal axis of the polynucleotide double strandat the state at which the charge separating group in the detectingsection is caused to be charge-separated depends on the pi-electronstack of aromatic rings of the bases forming nucleotides. Thepolynucleotide double strand loses electrical conductivity when thepi-electron stack temporarily loses its balance due to its molecularmotion (fluctuation of the molecular structure) when a particularprotein is bonded to the bonding section located at the end of adetecting unit, this molecular motion is subjected to the action of themass of the protein, and becomes slower relative to the case where theprotein is not bonded.

By way of example, electrical conductivities through a polynucleotidedouble strand in the cases of no protein is bonded and a protein isbonded are detected as responses with time as schematically shown inFIGS. 1A and 1B, respectively, because the molecular motion is morevigorous in the former case, and is slower in the latter case.

The effect of loss of electrical conductivity of the polynucleotidedouble strand structure may be larger by enhancing the fluctuation ofthe double strand by purposely introducing a crack in the polynucleotidedouble strand structure by, for example, providing discontinuity at oneor more locations somewhere along one nucleotide chain of the doublestrand. To provide the nucleotide chain with discontinuity, it is usefulto cut out part of a phosphate residue linking adjacent nucleotides toeach other. Alternatively, a structure containing a nucleotide chainhaving a discontinuity can be also obtained by combining one nucleotidechain with one or more short nucleotide chains, which have only part ofa sequence complementary to the sequence of the former nucleotide chain,to thereby spontaneously provide a polynucleotide double strand.

The electrode for picking up the change in electrical conductivity of oramount of transferred charge in a polynucleotide double strand modifiedby the bond of a protein is made from a material which is stable in asample solution. For the detection/determination of plural kinds ofproteins, an electrode is provided for each of the proteins, and one endof a polynucleotide double strand of the detecting section is connectedto each of the electrodes, with the detecting section being connectedto, at the other end, the bonding section formed of an antibody for eachof the proteins.

The connection of the polynucleotide double strand to the electrode canbe effected by linking a mercapto group introduced to one end of onenucleotide chain to the electrode through a spacer (—(CH₂)_(n)— or thelike is typically used). The polynucleotide double strand structure thusconnected to the electrode nearly linearly extends at a nearly constantangle to the surface of the electrode.

As components other than the detecting unit composing the proteindetecting device of the invention, i.e., the standard electrode,reference electrode, sample container and measuring unit, those used forthe measurement of a conductivity of a substance in a sample solutioncan be used.

The protein detecting device according to the second aspect of theinvention will now be described. This device uses, as the proteindetecting unit, a unit having a bonding section, which has propertiesfor specifically bonding to a protein to be detected, a sensing sectionmade up of a polynucleotide double strand and a fluorescent pigmentgroup, a detecting section for detecting the bonding of the protein tobe detected to the bonding section, the detecting section being made upof a quenching pigment group, and a controlling section for setting theconformation of the sensing section at an initial state, the controllingunit being made up of a pair of electrodes.

The bonding section in this detecting unit may be similar to the bondingsection used in the detecting unit of the device of the first aspect ofthe invention.

The sensing section is formed as a nano-structure made up of apolynucleotide double strand and a fluorescent pigment group. One end ofthe polynucleotide double strand is linked to the bonding section asdescribed above, and the other end is connected to one of a pair ofelectrodes in the controlling section described later. The fluorescentpigment group may be added to the polynucleotide double strand bycovalent bonding, or may be contained within the structure of thepolynucleotide double strand (insertion between adjacent complementarymoieties (intercalation)), or may be incorporated within a nucleotidechain by replacing part thereof. The fluorescent pigment group isdesigned to be present at the vicinity of the end of the polynucleotidedouble strand for the bonding section.

The fluorescent pigment group is selected from substances which areexcited by light to generate fluorescence. Examples of the fluorescentpigment groups which can be preferably used in the invention includefluorescein maleimide, Cy3 (trade mark) and the like.

The detecting section is made up of a quenching pigment group fordetecting the bond of a protein to be detected to the bonding section,and the quenching pigment group is fixed to any support. In general, thequenching pigment group is fixed to an electrode, to which the sensingsection as described above is linked, of a pair of electrodes in thecontrolling section described below.

The quenching pigment group is selected from those causing effectiveoptical quenching for a fluorescent pigment group used. For example,when fluorescein maleimide or Cy3 (trade mark) is used as thefluorescent pigment group, D-damine B sulfonyl chloride or Cy5 (trademark) can be used, respectively. More preferably, a combination of afluorescent pigment group and a quenching pigment group, which providesa FRET (fluorescence resonance energy transfer) effect, is used.

The controlling section is made up of a pair of electrodes. One of theelectrodes (a first electrode) is connected to one end, which is notconnected to the bonding section, of the polynucleotide double strandforming the sensing section, and the other (a second electrode) ispositioned separately from the bonding section, sensing section anddetecting section.

It is known that when one end of the polynucleotide double strand isfixed to a first electrode, this electrode is immersed together with asecond electrode in an aqueous solution, and an electric field, e.g., analternating electric field, is applied to between these electrodes, thepolynucleotide double strand is linearly elongated in the direction ofthe electric field, and, when the electric field is eliminated, thestrand is spontaneously flocculated. The protein detecting device of theinvention utilizes this property of polynucleotide double strand, tothereby detect whether or not a protein to be detected is bonded to anantibody fixed to the end on the polynucleotide double strand.

When the detecting unit composed as described above is placed in asample solution containing a protein to be detected, and an electricfield is applied between the electrodes of the controlling section, thedouble strand will be in a state of the elongation in the direction ofthe electric field (initial state), as described above. When thedetecting unit in this state is irradiated with appropriate light froman exciting source, the fluorescent pigment group is excited andgenerates fluorescence. Thereafter, by removing the electric field, thedouble bond, which has been elongated, is flocculated. At this time, asthe end of the double strand structure of the sensing section, opposedto the end thereof having the fluorescent pigment group bonded, isconnected to the electrode to which the quenching pigment groups arefixed, the fluorescent pigment group is relatively close to thequenching pigment group and, consequently, a decrease in the intensityof fluorescence is observed. When the electric field is applied again,the double strand is elongated and, accordingly, the bonding sectionregains the linearly elongated conformation at the initial state. It isalso possible to designate the conformation of flocculated double strandstructure for the initial state. In this case, the intensity offluorescence is decreased in the initial state, and the double strandstructure is elongated by the application of electric field, resultingin the increase in the intensity of fluorescence.

When a protein to be detected is present in a sample solution, theprotein is bonded to an antibody at the bonding section. As a result,the mass of the end of the double strand structure is increased and,citing an example of the double strand structure elongated at theinitial state, a time required to complete the spontaneous flocculationafter the removal of electric field, i.e., a time taken for theintensity of fluorescence to be reduced to a certain level, isincreased, leading to the variation in a time constant. Accordingly, bydetecting the difference in time constant during the increase ordecrease in the intensity of fluorescence at the initial state to acertain level, it can be detected whether or not a protein is bonded tothe bonding section. An extent to which the time is increased (ordecreased) is varied depending on the amount of protein present in asample solution, so that, by knowing the extent, the amount of proteinin the sample solution can be determined, and the type of protein can beidentified depending on from what double strand, among an array ofdouble strands, the signal comes.

By way of example, respective responses observed as the decrease in theintensity of fluorescence in the cases of a protein bonded to and notbonded to the bonding section are schematically shown in FIGS. 2A and2B.

As components other than the detecting unit composing the proteindetecting device of the invention, i.e., the container for a samplesolution, electric power supply, source of exciting light, and unit formeasuring fluorescence, those commonly available can be used.

The protein detecting device according to the third aspect of theinvention will now be described. This device uses, as the proteindetecting unit, a unit having a bonding section, which has propertiesfor specifically bonding to a protein to be detected, a detectingsection for detecting the bonding of the protein to be detected to thebonding section, the detecting section being made up of a polynucleotidedouble strand and a light emitting group, and an electrode section toreceive exciting electrons from the light emitting group for thequenching of the light emitting group excited by light.

The bonding section in this detecting unit may be similar to the bondingsection of the detecting unit used in the device of the first aspect asearlier described.

The detecting section is formed as a nano-structure made up of apolynucleotide double strand and a light emitting group. As in thedetecting unit of the first aspect, one end of the polynucleotide doublestrand is connected to the bonding section, and another end is connectedto the electrode section.

As the light emitting group, a substance which is excited by irradiatedexciting light and, in turn, emits light is used. As the light emittinggroup, a substance, such as a fluorescent pigment group as earlierreferred to in the description of the device of the second aspect, canbe used.

Also in the third aspect, the light emitting group may be added to thepolynucleotide double strand by covalent bonding, or may be containedwithin the structure of the polynucleotide double strand (insertedbetween adjacent complementary moieties (intercalation)), or may beincorporated within a nucleotide chain by replacing a part thereof.

The electrode section used in the detecting unit of the device of thethird aspect and the connection of the polynucleotide double strandthereto are as set out in the description of the detecting unit of thedevice of the first aspect.

In the device of the third aspect, the light emitting group is at thefree end of the elongated polynucleotide double strand which is liableto fluctuate, so that it spontaneously fluctuates. Accordingly, in thisdevice, the detection of protein is effected by the use of, for example,a phenomenon that the emission from the light emission group of thedetecting section by the irradiation of exciting light fluctuatesdepending on the fluctuation of the polynucleotide double strand. Thebonding of a protein to be detected to the bonding section leads to anincrease in mass of the bonding section, and a slower fluctuation of thepolynucleotide double strand of the detecting section compared to whenthe protein is not bonded. In consequence, the slower fluctuation isobserved as slower fluctuation of the emission from the light emittinggroup and, using this, the bonding of the protein can be detected. Bypreviously preparing a calibrated curve, quantitative determination ofthe protein is also possible.

More specifically, when the light emitting group is irradiated withexciting light, the electron of a light emitting group is excited, andfluorescence is observed when the excited electron goes back to anenergy level lower than that at the excited state. On the other hand,the emission is prevented, and so-called quenching occurs, when theenergy of the excited electron is lost outside the light emitting groupdue to, for example, overlap of electron orbits. If a substance forabsorbing a positive or a negative charge is then provided outside thelight emitting group, one of charges is lost from the light emittinggroup, resulting in charge separation (the development of charges). Whenall complementary base pairs are perfect in the polynucleotide doublestrand of the detecting section in the invention, the pi-electron orbitsin the aromatic rings of adjacent bases forming the nucleotides overlapwith each other, and form a so-called pi-electron stack to therebyexhibit electrical conductivity in the longitudinal direction of thestrand. Migration of a high energy site or electric charge generated bythe charge separation through the overlap of electron orbits makes thedevelopment of quenching easier. If the polynucleotide is then connectedat one end to an electrode, the high energy site or electric charge lostfrom the light emitting group moves towards the electrodes as anelectric current.

The polynucleotide double strand anchored to the electrode has naturalfluctuation of structure due to thermal motion of molecules. When acrack (also called “nick”) is introduced to one of the complementary twochains of polynucleotide, the fluctuation is enhanced, so that twoconditions occur, the pi-electron stack being temporally broken up inone condition, and being restored in the other. Based on this, themovement of the high energy site or charge through the pi-electron stackis temporally prevented, which means that, with the molecular motion ofpolynucleotide, the loss and restoration of the quenching phenomenon arecontinuatively observed, and during which the intensity of emissionvaries corresponding to the motion (fluctuation) of the polynucleotidedouble strand. The fluctuation of the polynucleotide double strand isslow when the protein to be detected is bonded to the bonding section,and is fast when it is not bonded, as described above, so that it can beknown, by the change in the intensity of emission, whether or not theprotein is bonded to the bonding section.

FIG. 3A represents a graph schematically showing, as a function of time,the intensity of emission from a light emitting group and the amount ofcurrent running from the light emitting group to an electrode through apolynucleotide in the case where an objective protein is not bonded tothe bonding section, and FIG. 3B represents a similar graph in the casewhere the objective protein is bonded to the bonding section. In thecase where the protein is bonded, the bonding section has an increasedmass, and the motion of the polynucleotide double strand will be slowercompared to the case where the protein is not bonded. As a result, thetime of quenching due to movement of the high energy site or electriccharge through the pi-electron stack (during which the intensity ofemission is lowered or not observed, and the high energy site or chargetravels the pi-electron stack), and the time of emission during whichthe excited electron does not travel the pi-electron stack (and duringwhich the intensity of emission is increased or observed), will belonger compared to the case where the protein is not bonded to thebonding section (the fluctuations of the intensity of emission and thecurrent are mitigated).

As components other than the detecting unit composing the device of thethird aspect of the invention, i.e., the container for sample solution,a unit for measuring emission, and a source of exciting light in thecase of exciting the light emitting group by light, those commonlyavailable can be used.

In the biopolymer detecting device of the invention, a member having abonding site for detecting a biopolymer to be detected is used, themember being made up of a molecule having an affinity to the biopolymer,such as a protein, or a compound having interaction with the biopolymerto be detected. Representatives of the molecules or compounds arevarious proteins, including antibodies, RNAS, oligonucleotides and thelike. It is also possible to use a complex of a combination of them asthe member having the bonding site for detecting the biopolymer. By wayof example, a complex of a combination of a protein and a nucleotide ora complex of a combination of an antibody and a nucleotide may bereferred to.

The molecule or compound is fixed to an electrode of the detectingdevice of the invention. This can be effected by allowing a solutioncontaining a certain molecule or compound to be contacted with theelectrode for a predetermined time.

The molecule or compound may be fixed directly to the electrode (or aconductor layer of a stack, which is contacted directly with the samplesolution, when the electrode incorporates a stack of layers ofdielectric and conductor as described hereinafter), or may be fixedindirectly to the electrode through an appropriate ligand, such asthioether (—S—) group, or through a compound, such as a nucleotide. Thefixing of the molecule or compound to the electrode (or the conductorlayer) can be also effected using a material called a carbon nanotube orcarbon nanofiber. The carbon nanotube or nanofiber can be formed bydisposing a catalyst (made of, e.g., Ni or the like) on the electrode(or the conductor layer) to which the molecule or compound is to befixed, and perpendicularly growing nanotubes or nanofibers from thesurface of the electrode (or the conductor layer) by thermal CVD orplasma-enhanced CVD. A portion of the five-member rings at the end ofthe formed carbon nanotube or nanofiber can be readily chemicallymodified. Using this property, a certain molecule or compound can bejoined to the end of the carbon nanotube or nanofiber. The carbonnanotube or nanofiber is a hard material, so that a molecule or compoundbonded to the electrode (or the conductor layer) through such a materialis more strongly fixed thereto, which is favorable for, for example, acase where a sample solution having a high viscosity is used (the fixedmolecule or compound is subjected to a significant force when ahigh-viscosity solution is introduced to between the electrodes or isdischarged after the measurement).

The electrode is made from a conductive material and, in general, ametallic material (for example, aluminum, copper or gold) can be usedtherefor. A pair of electrodes is arranged so as to sandwichtherebetween a sample solution-holding section for holding a samplesolution containing a biopolymer to be detected, to thereby form a typeof capacitor. In this capacitor structure, at least one electrode can beformed so as to include a dielectric layer. When both electrodes areformed so as to include a dielectric layer, at least one electrode isconfigured so as to be indirectly contacted with a sample solution notthrough the dielectric layer but through the conductor layer. Forexample, when the electrode includes one dielectric layer, it isconfigured to have a stack structure of conductor layer/dielectriclayer/conductor layer, and the member having the bonding site fordetecting a biopolymer to be detected is fixed to the conductor layerwhich will be in contact with a sample solution. One electrode maycontain a plurality of dielectric layers and, in this case, a conductorlayer is interposed between adjacent dielectric layers using such anelectrode including a dielectric layer or layers is useful to change acapacitance of a capacitor having a sample solution sandwiched betweenthe electrodes thereof, to thereby change the sensitivity of a device.The dielectric layer may be formed using, for example, SiO₂, SiON,Si₃N4, carbon-based dielectric materials, tantalum oxide and the like.In the case of a stack structure containing a plurality of conductorlayers, the conductor layers may be formed from the same material, ormay be formed from different materials. Similarly, in the case of astack structure containing a plurality of dielectric layers, they may bealso formed from the same material, or they may be formed from differentmaterials.

The biopolymer detecting device of the invention uses one or more pairsof capacitor electrodes to which a molecule having an affinity to aspecific biopolymer, such as a protein, to be detected, or a compoundhaving interaction with the biopolymer to be detected, anddetects/determines the biopolymer present in a sample solution based onthe change in signal resulted from the change in capacitance between thecapacitor electrodes caused by the bond of the biopolymer to theelectrodes. For the detection/determination, a calibration curve,prepared in advance, can be used. The detection and determination of thebiopolymer to be detected are possible even when the detecting devicehas only one pair of electrodes. If two or more pairs of electrodes towhich a molecule or compound useful for the detection of a biopolymer isfixed, a plurality of types of biopolymers present in a sample solutioncan be detected/determined together, based on the correspondencerelationship between a signal developed by the bond of the biopolymer tothe electrode and the location of the electrode developing the signal.

An electrical signal, as a signal that changes in accordance with thechange in capacitance between the capacitor electrodes, caused by thebond of the biopolymer to be detected to the electrode, is processed byan electric circuit contained in a detecting device. Specifically, theelectrical signal is detected, herein, as the change in the amount ofelectric charge (interelectrode capacitance) accumulated between theelectrodes, or as the difference in current passing between an electrodeof one pair of electrodes having the site capable of being bonded to thebiopolymer to be detected and an electrode of another pair of referenceelectrodes, as a result of the difference in the amount of accumulatedcharge between one pair of electrodes and another pair of electrodes.For example, the change in electric charge is measured by a coulometer,and the current is measured by an ammeter. Alternatively, themeasurement may be effected using a transistor, a device using an MISstructure, a device using a p-n junction, a device having a Schottkyjunction, or a combination thereof. Such a measuring means may beincluded in the detecting device of the invention, or may be set upoutside the device. The electric circuit for processing electricalsignals in the detecting device of the invention is constructed so as tocontain such a measuring means in the former case, and is designated forthe connection (electrical connection) with an external measuringinstrument in the latter case, with the measurement itself beingexternally carried out (in this case, the processing in the electriccircuit of the detecting device represents the transmission of theelectric signal to the exterior).

The biopolymer detecting device of the invention can be provided withterminals (lead electrodes) for connecting its electric circuit to anexternal electrical circuit. In the case of the device in which thechange in the amount of electric charge accumulated between theelectrodes is measured, the device having only one terminal of thesimplest embodiment makes in possible to carry out the measurement by anexternal ammeter which is grounded.

The device of the invention needs to have a sample solution-holdingsection for holding a sample solution containing a biopolymer betweenthe electrodes during the measurement. The sample-solution holdingsection may be a gap portion between the electrodes of a capacitorstructure. Fluid channels, such as those for feeding a sample solutionto the holding section and for discharging the sample solution from theholding section, may be connected to the holding section.

Preferably, the devices of the invention are fabricated in the samesubstrate utilizing the techniques of photolithography and filmformation used in the manufacture of semiconductor devices and the like.As the substrate, a substrate of silicon or sapphire can be used.

The biopolymer detecting device according to the invention represents aso-called protein chip, and is designed to detect, for example, thedecline or enhance of part of a series of protein interaction networkfunction starting from insulin receptor and ending with glycogencatabolic enzyme in the case where hepatocyte having diabetes switchesthe intracellular metabolism of glycogen depending on a state of insulinacceptance. By the use of this technique, it becomes possible to knowthe population of proteins, including a so-called posttranslationalmodification such as a phosphorylation or an addition of a sugar chain.Consequently, instead of detecting diabetes mellitus on the basis ofsymptom as before, it becomes possible to know that the decline infunction of a certain protein pertaining to the interaction networkcauses incompetence of glucose metabolism, and appropriate diagnosis andcure corresponding to the cause of disfunction, as well as verificationof a therapeutic result, become possible.

Of course, a similar technique can be applied to the entirety ofmultifactorial disease, such as hypertension, hyperlipidemia or thelike, in addition to diabetes mellitus.

EXAMPLES

The invention will now be described referring to the following examples,although the invention is not to limited to these examples.

Example 1

As schematically shown in FIG. 4, a one-chain polynucleotide 11 a, toone end of which a mercapto group was introduced through a spacer, wassynthesized, and was hybridized with a complementary one-chainpolynucleotide 11 b, which was similarly synthesized, to form apolynucleotide double strand structure 11, which was then reacted with apolished gold electrode 13 at room temperature for 24 hours to be bondedto the gold electrode 13.

Using a solution in which iron porphyrin, as a charge separating group15 causing charge separation, was dissolved, the charge separating group15 was absorbed to (intercalated in) the pi-electron stack of thepolynucleotide-double-strand structure 11 on the gold electrode 13.Alternatively, a group derived from such a charge separating group maybe covalent-bonded to the polynucleotide chain, or part of a nucleotidebase may be substituted with a group derived from such a pigment.Subsequently, a Fab fragment 17 of a monoclonal immunoglobulin IgG wasfixed to the end of the polynucleotide chain.

As shown in FIG. 4, a detecting unit 21 thus fabricated was formed of anelectrode section 23, a detecting section 25, and a bonding section 27.The antibody 17 of the bonding section 27 is specifically bonded only toa particular protein 31 to be detected.

The electrode 13 is, in general, positioned on a support which is notshown. By arranging, in a single detecting unit, a plurality ofelectrodes 13 in the form of an array, and bonding different antibodiesto the bonding sections corresponding to different electrodes, itbecomes possible to detect/determine a plurality of proteins by thesingle detecting unit.

The detecting unit 21 as fabricated above was immersed in an aqueoussolution containing ferrocyanide ion as a charge acceptor, and a currentrunning through the polynucleotide double strand structure 11 wasmeasured by a three-electrode method using a reference electrode (notshown). Subsequently, using an aqueous solution containing a protein tobe detected in addition to the charge acceptor, a similar measurementwas performed.

The conductivity developed by the polynucleotide double strand in thelongitudinal direction of the strand by the pi-electron stack of thearomatic rings of bases composing the nucleotides was lost in acondition that the stack of pi-electron was temporarily broken up bymolecular motion of the double strand structure. It was observed thatthe temporary loss of conductivity was made preeminent particularly bypurposely introducing a crack or cracks in the double strand, as shownin FIG. 4.

In addition, when a solution containing no proteins was used for themeasurement, no differences were observed in appearance of the loss andrestoration of the conductivity measured for the respective goldelectrodes. In contrast, when the solution containing proteins was used,it was observed that the loss and restoration of the conductivity wasslow for the electrodes corresponding to the bonding sections to whichthe protein was bonded, and patterns of the loss and restoration of theconductivity observed for those gold electrodes were different from eachother corresponding to the molecular weights of the bonded proteins.Fluctuation of the conductivity occurred at a high frequency for thenucleotide chain to which no protein was bonded, and the fluctuation wasmitigated when a protein was bonded to the nucleotide chain to increasethe mass thereof, the mitigation of the fluctuation being more enhancedas the mass of the protein bonded became larger.

Thus, in the invention, the determination of a protein to be detectedcan be made by detecting a phenomenon of the fluctuation of conductivitydeveloped by a polynucleotide double strand being mitigated by theprotein bonded thereto. By the use of a plurality of electrodes, and theuse of different antibodies for different bonding sections correspondingthe electrodes, it also becomes possible to identify and detect unknownkinds of protein.

The sensitivity for detecting a protein is varied depending on themolecular weight of a protein bonded to the bonding section, and is alsovaried depending on a bonding constant of the protein with a monoclonalIgG antibody. Accordingly, by the use of a plurality of measuringelectrodes for the same protein, and providing, corresponding to therespective electrodes, bonding sections of monoclonal antibodies havingbonding constants for the protein which are different from each other,measurement can be performed over a wide range of the concentration ofprotein.

The entire construction of a measuring apparatus using the proteindetecting device illustrated in this example is schematically shown inFIG. 5. The measuring apparatus shown in the drawing uses a proteindetecting device 40 comprising a sample container 45 which contains adetecting unit 21 made up of a support 22, the electrode section 23, thedetecting section 25 and bonding section 27, a standard electrode 41,and a standard electrode 43, and further including a measuring unit 47which is connected to the respective electrodes 23, 41, 43. A samplesolution 48 containing a protein to be detected is added to the samplecontainer 45. If the detecting section 25 is of a type of chargeseparating group generating an electron and an positive hole upon lightexcitation, there is provided an exciting light source 49. The measuringunit 47 of the protein detecting device 40 is preferably connected to adata processing device 51 for processing measured data, the dataprocessing device 51 being accompanied with a device 53 for displayingmeasured results and a memory storage or storages 55, 55′ for storingthe arrangement of an array, calibrated values for the detecting unit 21and the like.

Example 2

A one-chain polynucleotide, to the 5′ end of which a mercapto group wasintroduced through a spacer, was synthesized, and was hybridized with aone-chain polynucleotide, which had a complementary sequence and the 5′end to which a fluorescent pigment (Cy3 (trade mark)) was introduced, toform a polynucleotide double strand structure, which was then reactedwith a polished gold electrode positioned on a support at roomtemperature for 24 hours to be bonded to the gold electrode. It ispreferable that the degree of polymerization of the polynucleotide chainis such that it has 12 to 100 monomer-residues. The mercapto group andthe fluorescent pigment may be introduced to the ends of one of theone-chain polynucleotides, respectively, or may be introduced to the 3′ends of both the one-chain polynucleotides.

The polynucleotide double strand structures were fixed in a circulararea of 10 micrometers, around which a spacer area having nopolynucleotide double strands fixed was provided. As a quenching pigmentproviding effective quenching for the fluorescent pigment introduced tothe polynucleotide chain, Cy5 (trade mark) was fixed onto the surface ofthe gold electrode. In addition, a Fab fragment of monoclonalimmunoglobulin IgG was fixed to the end of the polynucleotide chain.Herein, Fab fragments which were different in specificity were fixed tothe respective groups of polynucleotides separated from each other bythe spacer area.

The detecting unit thus fabricated is schematically shown in FIG. 6. Thesupport, gold electrode, polynucleotide double strand (which is depictedby a single line for simplification), fluorescent pigment, Fab fragment,protein, and quenching pigment are identified by reference numerals 61,63, 65, 67, 69, 71, and 73, respectively.

The detecting unit was then put into contact with a sample solutioncontaining a protein to be detected, and was allowed to stand at roomtemperature for a sufficient time to form a bond of the Fab fragment 69and the protein 71, as shown in FIG. 6.

The detecting unit having the protein thus bonded was immersed in anaqueous solution, as also shown in FIG. 6, and an AC field was appliedto the polynucleotide double strand structures 65 by a two-electrodemethod (alternatively, a three-electrode method may be used) using a ACpower supply 77, waiting until the fluorescent pigment 67 on thepolynucleotide double strand was excited by a UV lamp 79 to generatefluorescence and the intensity of fluorescence-became steady.Subsequently, the AC field was removed, and after the establishment of asteady state, rates of decrease in the intensity of fluorescence weremeasured for the respective groups of polynucleotide double strandstructure.

The left polynucleotide double strand structure 65 schematicallydepicted in FIG. 6 represents one at an electric field applied state,and the right double strand structure 65 represents one at a no electricfield state (no electric filed applied state). For the double strandstructure 65, having been linearly elongated at the electric fieldapplied state, the polynucleotide double strand is spontaneouslyflocculated upon disappearance of the electric field, and thefluorescent pigment 67 at the end of the double strand structure 65comes close to the quenching pigment 73 on the surface of the goldelectrode, as shown in the right side of FIG. 6, whereby a decrease inthe intensity of fluorescence is observed.

When the protein to be detected present in the sample solution is bondedto the Fab fragment, the mass at the end of the polynucleotide doublestrand is increased, resulting in the increase in time taken for thespontaneous flocculation, i.e., time taken for the decrease in theintensity of fluorescent. The extent by which the time is increased isvaried depending on the amount of protein present in the samplesolution, so that, by knowing the extent, the amount of protein in thesample solution can be measured, and the type of protein can beidentified depending on what polynucleotide strand the signal comesfrom.

Also, in this example, the sensitivity for detecting a protein is varieddepending on the molecular weight of a protein bonded to the bondingsection, and is also varied depending on a bonding constant of theprotein with a monoclonal antibody. Accordingly, by arranging, in theform of an array, several sensing sections using monoclonal antibodieshaving different bonding constants for the same protein, measurement canbe performed over a wide range of the concentration of protein.

The entire construction of a measuring apparatus using the proteindetecting device illustrated in this example is schematically shown inFIG. 7. The measuring apparatus shown in the drawing uses a proteindetecting device 110 comprising a detecting unit 92 made up of a support80, a gold electrode 82, which is one electrode of a controllingsection, a detecting section 84 of a quenching pigment, a sensingsection 86 of polynucleotide double strand structures and a fluorescentpigment, a bonding section 88 of a Fab fragment and another electrode 90of the controlling section, and a sample container 94 in which thedetecting unit 92 is contained, and further including a power supply 96connected to the respective electrodes 82, 90, an exciting light source98, and a fluorescence measuring unit 100. A sample solution 102containing a protein to be detected is added in the sample container 94.The measuring unit 100 of the protein detecting device 110 is preferablyconnected to a data processing device 122 for processing measured data,the data processing device 122 being accompanied with a device 124 fordisplaying measured results and a memory storage or storages 126, 126′for storing the arrangement of an array, calibrated values for thedetecting unit 92 and the like.

Example 3

A detecting unit was fabricated as in Example 1, except that Cy3 or FITCwas used as an light emitting group in place of the charge separatinggroup used in Example 1.

A one-chain polynucleotide, to one end of which a mercapto group wasintroduced through a spacer, was synthesized and was hybridized with acomplementary one-chain polynucleotide 11 b, which was similarlysynthesized, to form a polynucleotide double strand structure, which wasthen reacted with a polished gold electrode at room temperature for 24hours to be bonded to the gold electrode.

Using a solution in which a light emitting group was dissolved, thelight emitting group was intercalated in the pi-electron stack of thepolynucleotide double strand on the gold electrode. Alternatively, anucleotide base may be replaced by a derivative of the light emittinggroup. Subsequently, a Fab fragment of a monoclonal immunoglobulin IgGwas fixed to the end of the polynucleotide chain.

A detecting unit thus fabricated was as shown in FIG. 4, and was similarto the detecting unit fabricated in Example 1, except that ironporphyrin was used as a charge separating group 15 in Example 1, whereasthe Cy3 was used herein as the light emitting group 15. Thus, thedetecting unit 21 in this example was also formed of an electrodesection 23, a detecting section 25, and a bonding section 27. Also, theantibody 17 of the bonding section 27 is specifically bonded only to aparticular protein 31 to be detected.

The fabricated detecting unit 21 (FIG. 4) was immersed in a samplesolution containing no protein to be detected, and was irradiated withexciting light to make the light emitting group emit light. It wasconfirmed that the intensity of emission was significantly increased inthe condition that the electrical conductivity of the pi-electron stackof the polynucleotide double strand was temporally lost by molecularmotion (fluctuation of the intensity of emission was observed). Inaddition, in a detecting unit in which a crack was purposely introducedin the polynucleotide double strand, it was observed that thefluctuation of the intensity of emission was more significant.

Subsequently, the detecting unit was immersed in a sample solutioncontaining a protein 31 (FIG. 4) to be detected, and was allowed tostand at room temperature for a sufficient time for the protein 31 to bebonded to the Fab fragment 17, after which a similar experiment wascarried out by irradiating the detecting unit with exciting light. Inthis case, the fluctuation of the intensity of emission was slowercompared to the case where the protein was not bonded, due to theincrease in mass in the bonding section of the detecting unit by thebond of the protein thereto.

Thus, the detection of a protein to be detected becomes possible bydetecting a phenomenon of the fluctuation of conductivity developed by apolynucleotide double strand being mitigated by the protein bondedthereto, as the fluctuation of the intensity of emission from the lightemitting group added to the polynucleotide double strand. In addition,use of a calibration curve makes the determination of the proteinpossible.

Sensitivity for detecting a protein is varied depending on the molecularweight of a protein bonded to the bonding section, and is also varieddepending on a bonding constant of the protein with a monoclonal IgGantibody. Thus, by the use of a plurality of measuring electrodes forthe same protein, and providing, corresponding to the respectiveelectrodes, bonding sections of monoclonal antibodies having bondingconstants for the protein which are different from each other,measurement can be performed over a wide range of the concentration ofprotein.

The entire construction of a measuring apparatus using the proteindetecting device illustrated in this example is schematically shown inFIG. 8. The measuring apparatus shown in the drawing uses a proteindetecting device 200 comprising a detecting unit 21 made up of a support22, an electrode section 23, a detecting section 25 and a bondingsection 27, a sample container 201 in which the detecting unit 21 iscontained, an exciting light source 202, and a measuring unit 203. Asample solution 204 containing a protein to be detected is added in thesample container 201. The measuring unit 203 of the protein detectingdevice 200 is preferably connected to a data processing device 211 forprocessing measured data, the data processing device 211 beingaccompanied by a device 212 for displaying measured results and a memorystorage or storages 213, 213 for storing the arrangement of an array,calibrated values for the detecting unit 21 and the like. If a lightemitting group, which emits light upon the application of bias, is used,the exciting light source 202 can be omitted.

In this apparatus, the electrode section 23 of the detecting unit 21 isconnected to the measuring unit 203. In the case where a light emittinggroup of a type of emitting light upon the application of bias is usedin place of the light emitting group emitting upon the irradiation withexciting light used in this example, it is possible to apply bias to thelight emitting group through the electrode section 23. When the currentrunning from the light emitting group to the electrode section 23through the polynucleotide double strand is to be detected, in additionto the intensity of emission, the measuring unit 203 can include anammeter (not shown) to thereby carry out the current measurement.

Example 4

This example describes the production of a biopolymer detecting device.

As shown in FIG. 9A, a metallic layer made up of films of Ti, Pt, Au, Ptand Ti formed, in this order, on a silicon substrate 10 was patterned toform wiring lines 12. Of the drawings referred to in this example, onlyFIG. 9A is a top view, and the others are shown in cross-sections. Inaddition, the wiring lines 12 are depicted only in FIG. 9A, forsimplicity. Also, although a number of devices were simultaneouslyfabricated on a common silicon substrate, the drawings (FIGS. 9 and 10)associated with this example illustrate only a single samplesolution-holding section and the vicinity thereof, for simplicity.

As shown in FIG. 9B, an SiO₂ film 14 (1 micrometer thick) was formed onthe surface of the silicon substrate 10 having the wiring lines formed,and was patterned by dry etching using a resist pattern 16 formedthereon as a mask, to create trenches 18. Subsequently, an Au film (notshown) was formed on the entire surface of the substrate, and the resistpattern 16 and the Au film thereon were removed by a lift-off process,to thereby fill the trenches 18 (FIG. 9B) in the SiO₂ film 14 with Auconductor 20, as shown in FIG. 9C. The Au conductor 20 was designed tobe located on one end 12 a (FIG. 9A) of the wiring line 12 on thesilicon substrate 10, although the wiring lines 12 are not shown in FIG.9B and the following drawings.

As shown in FIG. 9D, an additional SiO₂ film 22 (1 micrometer thick) wasthen formed on the SiO₂ film 14, and the SiO₂ films 22 and 14 weresuccessively dry etched using a resist pattern 24 formed on theadditional SiO₂ film 22 as a mask, to thereby form trenches 26.Subsequently, an Si₃N₄ film (not shown) was formed on the entire surfaceof the substrate, and the resist pattern 24 and the Si₃N₄ film thereonwere removed by a lift-off process, to thereby fill the trenches 26(FIG. 9D) with Si₃N₄ dielectric 28, as shown in FIG. 9E. The Si₃N₄dielectric 28 thus filled had a height approximately equivalent to athickness of the underlayer of SiO₂ film 14.

Subsequently, as shown in FIG. 9F, a resist pattern 30 was formed so asto cover the upper layer of SiO₂ film 22, and was used as a mask forsuccessive dry etching of the SiO₂ films 22 and 14, to thereby form atrench 32, which serves as a sample solution-holding section of adevice, and at both sides of which the Au conductor 20 was exposed.

After peeling the resist pattern 30, the trench 32 was filled with asolution containing an oligonucleotide, and the oligonucleotide (notshown) was reacted with the Au conductor 20 at room temperature for 24hours to be bonded thereto, and to provide a biopolymer detecting device40 comprising a sample solution-holding section 38 sandwiched bycapacitors 36 a and 36 b, as shown in FIG. 9G.

Portion of the sample solution-holding section 38 of the device isenlarged and shown in FIG. 10. A number of oligonucleotides 42 arebonded at one end to the surface of the Au conductor exposed at bothsides of the sample solution-holding section 38.

Example 5

Following the procedure described in Example 4, a biopolymer detectingdevice 100, as shown in FIG. 11, was made. In the device described inExample 4, the electrodes at both sides of the sample solution-holdingsection had a capacitor structure made up of the two Au films and theSi₃N₄ dielectric film therebetween, whereas in the device 100 of thisexample having two sample solution-holding sections 102, 102′, anelectrode 104 (104′) at one side of each of the sample solution-holdingsections 102, 102′ forms a capacitor 112 (112′) made up of two Auconductor layer 108 a and 108 b (108 a′ and 108 b′) and an Si₃N₄dielectric layer 110 a (110 a′) interposed therebetween, and anelectrode 106 (106′) opposed to the electrode 104 (104′) and sandwichingthe sample solution-holding section therebetween is made up of an Auconductor layer 108 c (108 c′) and an Si₃N₄ dielectric layer 110 b (110b′), and forms a capacitor 114 (114′), which is connected in series tothe capacitor 112 (112′), together with the Au conductor layer 108 b(108 b′), which is opposed to the layer 110 b (110 b′) and sandwichesthe sample solution-holding section 102 (102′) therebetween. For theconnection with an external circuit, lead electrodes 120, 122, 120′,122′ connected to the respective electrodes 104, 106, 104′, 106′ wereprovided.

Antibodies 132 were bonded to the Au conductor layers 108 b, 108 b′exposed at the sample solution-holding sections 102, 102′ by filling thesample solution-holding sections 102, 102′ with an antibody containingsolution and allowing it to stand at room temperature for 24 hours. Tofacilitate the filling of the sample solution-holding section 102, 102′with the antibody-containing solution and a sample solution to bemeasured, the device 100 of this example is provided with fluid channels116 a, 116 b, 116 a′, 116 b′, communicating with the samplesolution-holding sections 102, 102′. Using the fluid channels, thesample solution-holding sections 102, 102′ can not only be filled with asolution, but also allow the solution to pass therethrough. The samplesolution-holding section had a length and a width of about 1 millimeterand about 1 micrometer, respectively.

The measuring system used in this example is schematically shown in FIG.12. In the drawing, a detecting system 130 for the samplesolution-holding section 102 is shown at the left side, and a detectingsystem 130′ for the sample solution-holding section 102′ is shown at theright side, corresponding to the device shown in FIG. 11. Prior to theinitiation of measurement, antibodies 132 were bonded to the Auconductor layers 108 b, 108 b′ exposed at the sample solution-holdingsections 102, 102′.

The capacitors 112 and 112′ were allowed to accumulate electric chargeby applying a DC bias to between the electrodes 104 and 106 and theelectrodes 104′ and 106′ of the detecting systems 130 and 130′,respectively, using a DC bias source (not shown). The circuit forapplying the bias was then disconnected, and a current passing betweenthe lead electrodes 122 and 122′, when there was a difference betweenthe electrical charges accumulated in the respective electrodes 106 and106′, was measured using a galvanometer 140, as follows.

A sample containing no protein bonding to the antibody 132 was passedfrom the fluid channels 116 a, 116 a′ (FIG. 11) to the samplesolution-holding sections 102, 102′, and an equivalent value of DC biaswas applied to between the lead electrodes 120 and 122 and the leadelectrodes 120′ and 122′, after which the circuit for applying the biaswas disconnected. The galvanometer 140 was connected to between the leadelectrodes 122 and 122′ to measure a current passing between the leadelectrodes 122 and 122′, and it was confirmed that no current wasdetected.

Next, a sample containing a protein bonding to the antibody 132 waspassed through the sample solution-holding section 102, 102′, and anequivalent value of DC bias was applied to the lead electrodes 120 and122 and the lead electrodes 120′ and 122′, after which the circuit forapplying the bias was disconnected. The galvanometer 140 was connectedto between the lead electrodes 122 and 122′ to measure a current passingbetween the lead electrodes 122 and 122′, and it was confirmed that nocurrent was detected.

Subsequently, the sample containing no protein bonding to the antibody132 was passed through the sample solution-holding section 102 of thedetecting system 130, the sample containing the protein 134 bonding tothe antibody 132 (the conditions of solvent for this sample were thesame as those for the sample containing no protein) was passed throughthe sample solution-holding section 102′ of the detecting system 130′,and an equivalent value of DC bias was applied to between the leadelectrodes 120 and 122 and the lead electrodes 120′ and 122′, afterwhich the circuit for applying the bias was disconnected. When thegalvanometer 140 was connected to between the lead electrodes 122 and122′ to measure a current passing between the lead electrodes 122 and122′, a current could be detected.

Similar measurements were carried out using samples having variousconcentrations of protein 134 bonding to the antibody, and a calibrationcurve of the protein concentration relative to the current value wasobtained by plotting the protein concentrations and the current valuesmeasured.

FIG. 6 shows a flow chart indicating the procedure when thedetermination of a protein to be detected is carried out using acalibration curve.

As described, the invention makes it possible to specifically andconveniently detect/determine a biopolymer, such as a protein, withoutlabeling the biopolymer itself with, for example, fluorescence. Byimplementing the invention as a device in which multiple pairs ofelectrodes are arranged on the same substrate in the form of an array,it is also possible to apply the invention to proteome analysis in whichbiopolymers are assessed as a mass.

1-11. (canceled)
 12. A protein detecting device, which comprises: (1) adetecting unit having a bonding section, which has properties forspecifically bonding to a protein to be detected, a sensing section madeup of a polynucleotide double strand and a fluorescent pigment group, adetecting section for detecting the bonding of the protein to bedetected to the bonding section, the detecting section being made up ofa quenching pigment group, and a controlling section for setting theconformation of the sensing section at an initial state, the controllingunit being made up of a pair of electrodes, (2) a container for housingthe detecting unit, and containing a sample solutions comprising theprotein to be detected, (3) an electric power supply connected to theelectrodes of the controlling section, (4) a source of exciting lightfor exciting the fluorescent pigment group in the sensing section togenerate fluorescence, and (5) a unit for measuring the fluorescence.13. The protein detecting device of claim 12, wherein the bondingsection is formed of a substance selected from the group consisting ofantibodies which specifically bond to the protein to be detected orfragments of the antibodies, which are obtained by limitedly decomposingthe antibodies by a protease, and organic compounds and biopolymershaving an affinity to the protein to be detected.
 14. The proteindetecting device of claim 13, wherein the substance forming the bondingsection is fixed to the polynucleotide double strand of the detectingsection by directly connecting it to the polynucleotide double strand.15. The protein detecting device of claim 13, wherein the substanceforming the bonding section is connected to the polynucleotide doublestrand of the detecting section through a connecting moiety useful toconnect and fix the substance to the polynucleotide double strand. 16.The protein detecting device of claim 12, wherein the bonding section isformed of an IgG antibody.
 17. The protein detecting device of claim 12,wherein the bonding section is formed of a monoclonal immunoglobulin IgGantibody.
 18. The protein detecting device of claim 12, wherein thebonding section is formed of a Fab fragment of a monoclonalimmunoglobulin IgG antibody or a piece derived from the Fab fragment.19. The protein detecting device of claim 12, wherein the initial stateof the conformation of the sensing section is set by applying anelectric field to between the electrodes of the controlling section ofthe detecting unit, which is in a solution, to thereby elongate thepolynucleotide double strand, or by removing an electric field appliedto between the electrodes to thereby flocculate the polynucleotidedouble strand.
 20. The protein detecting device of claim 12, wherein itis detected whether or not the protein is bonded to the bonding section,by the difference in time constant during the decrease or increase inthe intensity of fluorescence emitted from the sensing section at theinitial state of conformation to a certain level.
 21. A proteindetecting device, which comprises: (1) a detecting unit having a bondingsection, which has properties for specifically bonding to a protein tobe detected, a detecting section for detecting the bonding of theprotein to be detected to the bonding section, the detecting sectionbeing made up of a polynucleotide double strand and a light emittinggroup, and an electrode section to the surface of which the detectionsection is anchored, (2) a container for housing the detecting unit, andcontaining a sample solutions comprising the protein to be detected, and(3) a unit for measuring emitted light.
 22. The protein detecting deviceof claim 21, wherein the light emitting group is a compound which emitslight by being excited by light.
 23. The protein detecting device ofclaim 21, wherein the charge separating group is added to thepolynucleotide double strand by covalent bonding, or is interposed inthe structure of the polynucleotide double strand, or is incorporatedwithin a nucleotide chain forming the polynucleotide double strand byreplacing a part thereof.
 24. The protein detecting device of claim 21,wherein the bonding section is formed of a substance selected from thegroup consisting of antibodies which specifically bond to the protein tobe detected or fragments of the antibodies, and organic compounds andbiopolymers having an affinity to the protein to be detected.
 25. Theprotein detecting device of claim 24, wherein the substance forming thebonding section is fixed to the polynucleotide double strand of thedetecting section by directly connecting it to the polynucleotide doublestrand.
 26. The protein detecting device of claim 24, wherein thesubstance forming the bonding section is connected to the polynucleotidedouble strand of the detecting section through a connecting moietyuseful to connect and fix the substance to the polynucleotide doublestrand.
 27. The protein detecting device of claim 21, wherein thebonding section is formed of an IgG antibody.
 28. The protein detectingdevice of claim 21, wherein the bonding section is formed of amonoclonal immunoglobulin IgG antibody.
 29. The protein detecting deviceof claim 21, wherein the bonding section is formed of a Fab fragment ofa monoclonal immunoglobulin IgG antibody or a piece derived from the Fabfragment.
 30. The protein detecting device of claim 21, wherein one ofthe polynucleotide chains of the polynucleotide double strand has adiscontinuity at one or more locations along the chain.
 31. The proteindetecting device of claim 21, which further comprises a means formeasuring the intensity of emission from the light emitting group. 32.The protein detecting device of claim 31, which further comprises ameans for measuring a current resulting from excited electrons of thelight emitting group being moved to the electrode section through api-electron stack in the polynucleotide double strand by quenching.33-60. (canceled)